Stylasterid corals build aragonite skeletons in undersaturated water despite low pH at the site of calcification

Anthropogenic carbon emissions are causing seawater pH to decline, yet the impact on marine calcifiers is uncertain. Scleractinian corals and coralline algae strongly elevate the pH of their calcifying fluid (CF) to promote calcification. Other organisms adopt less energetically demanding calcification approaches but restrict their habitat. Stylasterid corals occur widely (extending well below the carbonate saturation horizon) and precipitate both aragonite and high-Mg calcite, however, their mode of biocalcification and resilience to ocean acidification are unknown. Here we measure skeletal boron isotopes (δ11B), B/Ca, and U/Ca to provide the first assessment of pH and rate of seawater flushing of stylasterid CF. Remarkably, both aragonitic and high-Mg calcitic stylasterids have low δ11B values implying little modification of internal pH. Collectively, our results suggest stylasterids have low seawater exchange rates into the calcifying space or rely on organic molecule templating to facilitate calcification. Thus, despite occupying similar niches to Scleractinia, Stylasteridae exhibit highly contrasting biocalcification, calling into question their resilience to ocean acidification.

Anthropogenic carbon emissions are causing seawater pH to decline, yet the impact on marine calcifiers is uncertain. Scleractinian corals and coralline algae strongly elevate the pH of their calcifying fluid (CF) to promote calcification. Other organisms adopt less energetically demanding calcification approaches but restrict their habitat. Stylasterid corals occur widely (extending well below the carbonate saturation horizon) and precipitate both aragonite and high-Mg calcite, however, their mode of biocalcification and resilience to ocean acidification are unknown. Here we measure skeletal boron isotopes (δ 11 B), B/Ca, and U/Ca to provide the first assessment of pH and rate of seawater flushing of stylasterid CF. Remarkably, both aragonitic and high-Mg calcitic stylasterids have low δ 11 B values implying little modification of internal pH. Collectively, our results suggest stylasterids have low seawater exchange rates into the calcifying space or rely on organic molecule templating to facilitate calcification. Thus, despite occupying similar niches to Scleractinia, Stylasteridae exhibit highly contrasting biocalcification, calling into question their resilience to ocean acidification.
At current anthropogenic carbon emission rates, surface seawater pH is predicted to fall below 7.8, and carbonate saturation states (Ω = [Ca 2+ ] × [CO 3 2− ]/K * sp ) will greatly reduce before the end of this century 1 . Such largemagnitude and (geologically) rapid ocean acidification is expected to be a key stressor for marine calcifying organisms 2 , with many studies showing a reduction in stony coral health, abundance, and calcification rates under low seawater pH conditions (e.g. 3 ). Yet without detailed knowledge of coral biocalcification mechanisms and adaptive strategies the full impact of ocean acidification on these marine calcifiers remains unclear. Particular concerns have been raised as to the vulnerability of deep-sea corals that remain poorly studied and occupy niches that are likely to be at the limits of their carbonate saturation tolerance already 4,5 .
Mineralogy plays an important role in dictating the susceptibility of carbonate to dissolution 6,7 . Calcite is a relatively dissolution-resistant form of CaCO 3 , typically adopted by unicellular foraminifera 8 . Aragonite is a more soluble polymorph, yet many organisms, including scleractinian corals, use this mineral 9 . As more magnesium is incorporated into calcite (up to ~ 140 mmol/mol Mg/Ca) its solubility increases towards that of aragonite 7 . Many organisms calcify using this intermediate solubility "high-Mg" calcite including Isidiid gorgonian "bamboo" corals 10 and sea urchins 11 . As the Mg content of calcite increases further (> 140 mmol/mol Mg/Ca) the solubility of this high-Mg calcite falls below even that of aragonite. Organisms that adopt this more soluble mineral form to build their skeletons include crustose coralline algae 12 .
With a diverse array of carbonate minerals used by marine organisms, different calcification pathways are required to construct and maintain these skeletons. However, there have been comparatively few studies where microsensors or pH sensitive dyes have been placed within living biocalcifiers to monitor pH at the site of calcification as they require sophisticated laboratory culture setup and cannot be applied to coral specimens postmortem (e.g. [13][14][15] ). Skeletal geochemistry is therefore the go-to approach for understanding the mineralisation process, with the boron isotopic composition (δ 11 B; 11 B/ 10 B ratio relative to the standard NIST SRM 951 in ‰) of marine carbonates shown to be a powerful tool to assess pH at the site of calcification 16,17 .
Scleractinian corals and coralline algae adopt more dissolution-susceptible mineral forms, but calcify from fluids that are semi-restricted from ambient seawater and in which pH is biologically elevated to promote calcification. This "pH upregulation" varies by taxon, but in cold-water Scleractinia it is ~ 1 pH unit above ambient seawater 16 . This strong elevation of pH in the CF results in skeletal δ 11 B values > 8‰ higher than seawater borate values ( Fig. 1; high ∆δ 11 B; where ∆δ 11 B = δ 11 B carbonate -δ 11 B borate ; Fig. 2) 16,[18][19][20][21][22] . While photosynthetic coralline algae are restricted to the photic zone with its generally higher carbonate saturation states, the adaptation of pH upregulation has enabled azooxanthellate cold-water Scleractinia to live much deeper, even in undersaturated seawater conditions (Fig. 2 5 ). It has been suggested that biogenic aragonites that lack strong pH upregulation strategies (resulting in low ∆δ 11 B values) such as the benthic foraminifera Hoeglundina elegans have a more restricted habitat and only calcify when ambient seawater is oversaturated with respect to aragonite, in areas such as the shallow Bahamas platform (Fig. 2 23 ). Many studies have suggested that the increased buffering associated with pH upregulation will make Scleractinia resilient against moderate changes in ocean pH 16,24 , however others continue to highlight the potential vulnerability of these corals in the deep sea and question the energetic demands of this biocalcification strategy 4,5,25 .

Figure 1.
Coral δ 11 B plotted against δ 11 B of the borate ion in seawater (a function of pH; blue labels). Stylasterid corals (circles; this study) are compared to the well-characterised aragonitic scleractinian Desmophyllum dianthus 16,30,31 that strongly upregulate pH at the site of calcification. D. dianthus replicates are averages and one shallow water fjord sample from McCulloch et al. 16 was excluded because of high seawater pH variability at that site. Error bars denote the estimated uncertainty on seawater borate δ 11 B.  16,30,31 , high-Mg calcitic bamboo corals 32 , calcitic and aragonitic foraminifera 35 , and crustose coralline algae 18 26,27 . The majority of stylasterid species can be found below 50 m water depth making this an important, but poorly studied, deep-water coral family 26,27 . These Hydrozoan corals differ significantly from scleractinian corals in their ability to precipitate skeletons of either aragonite, high-Mg calcite, or in some cases, both mineral polymorphs 26 . Compared to Scleractinia, skeletal δ 18 O and δ 13 C in stylasterid corals are close to equilibrium with seawater 28 , however a positive covariation of these δ 18 O and δ 13 C ratios suggests that there may be at least some biological modification of the internal seawater carbonate chemistry 28 . Contrary to expectation, the stylasterid corals that deviate least from equilibrium δ 18 O and δ 13 C values were found to be aragonitic specimens (~ − 3‰) implying that there is less modification of internal carbonate chemistry than high-Mg calcitic specimens, despite their greater mineralogical vulnerability to dissolution 28 . While this may represent a mineralogical control on skeletal stable isotope values, little is known about how stylasterids biomineralize and are able to thrive in undersaturated seawater conditions.
To address the gap in understanding of stylasterid coral biocalcification, we present the first skeletal δ 11 B, B/ Ca, and U/Ca data obtained from stylasterid coral skeletons (Fig. 3). The samples in this study lived in seawater environments ranging from pH 7.86 to 8.04 (Ω Arag. 0.9 to 2.0). They comprise two genera of stylasterid corals (Stylaster and Errina) that represent all three modes of stylasterid skeletal mineralogy: (i) solely aragonite, (ii) both aragonite and high-Mg calcite, and (iii) solely high-Mg calcite 28,29 . We use the same stylasterid specimens for which skeletal stable oxygen and carbon isotopes have been previously characterised 28 for comparison with our carbonate system proxy results.

Results
Stylasterid boron isotopes. Replicate measurements of bulk stylasterid δ 11 B of the same individual were generally within 0.5‰ of each other (Fig. 1). Once δ 11 B replicates for each stylasterid specimen are averaged, mean δ 11 B values are typically within 1‰ of the estimated δ 11 B composition of borate in ambient seawater for each coral (Fig. 1). The only samples (excluding Stylaster ibericus) with mean δ 11 B values more than 1‰ higher than δ 11 B borate were the high-Mg calcitic E. laterorifa (δ 11 B = δ 11 B borate + ~ 1.5‰). We therefore find little appreciable difference in δ 11 B between high-Mg calcitic and aragonitic stylasterid corals that live in similar seawater pH conditions. The single Stylaster ibericus specimen in this study yielded anomalously high δ 11 B (and low B/Ca) compared to other aragonitic stylasterids. This is discussed in detail in the Supplementary Information; however, its inclusion here has little impact on our overall findings.

Stylasterid B/Ca and U/Ca. B/Ca replicate measurements made on individual pieces of stylasterid coral
were typically within 7% for each specimen. U/Ca measurements were considerably more variable between replicates, on average differing by 14% for both aragonitic and calcitic specimens. Mean B/Ca and U/Ca values for high-Mg calcitic specimens were generally lower (< 330 and < 110 µmol/mol respectively) than aragonitic stylasterids (average 918 and 392 µmol/mol respectively) (Figs. 4 and 5). B/Ca and U/Ca results for the mixed mineralogy species E. gracilis lie on a mixing line between pure aragonitic specimens and high-Mg calcitic specimens, though falling closest to the pure aragonites, consistent with their 92 to 96 weight percent aragonite content 28 (Figs. 4 and 5).
Where there were differences between B/Ca and δ 11 B duplicate measurements (i.e. > 0.2‰; n = 6; excluding S. ibericus), in all cases B/Ca was found to be negatively correlated with δ 11 B for both aragonitic and mixed mineralogy specimens (Fig. 4). U/Ca values in aragonitic stylasterids show little covariance with previous δ 18 O measurements in the same specimens ( Fig. 5A; 28 ), but positive covariance with skeletal δ 11 B measurements (Fig. 5B), and slight negative covariance with B/Ca (Fig. 5C). These generalised trends hold both between specimens and between sample replicates (Figs. 4 and 5).

Discussion
The most important result in this study is the low skeletal δ 11 B measured in all stylasterid corals ( Fig. 1) with no apparent offset from ambient seawater δ 11 B borate (low ∆δ 11 B; Fig. 2). These results are in stark contrast to high ∆δ 11 B values recorded in scleractinian corals 16,30,31 and coralline algae 18,21,22 known to strongly modify pH at the site of calcification to promote mineral growth (Fig. 2). Instead, the low ∆δ 11 B values we record in stylasterid corals are more similar to bamboo corals 32 , sea urchins 11,33 , bivalves 33,34 , and unicellular foraminifera (e.g. Cibicidoides wuellerstorfi or H. elegans) 35 (Fig. 2). However, many of these organisms are composed of dissolutionresistant mineral forms (e.g. calcite). Stylasterids on the other hand, while not the first aragonitic taxon to exhibit low skeletal δ 11 B (e.g. H. elegans), are the first to show this feature whilst living at depths close to and below the carbonate saturation horizon 36 . Assuming that stylasterid δ 11 B reflects the pH of the CF according to the abiotic relationship between the borate ion and pH (Eq. (4); Methods; e.g. 16,17 ), these δ 11 B results suggest no modification of internal seawater pH in stylasterids. Furthermore, application of a physiochemical biocalcification model  47 , and crustose coralline algae (CCA 18,21 ). Individual analyses from the same specimens are connected by solid trend lines (2 analyses or more). Dashed trendline shows the covariance between all aragonitic stylasterid measurements (p < 0.01). Blue arrow shows theoretical mixing line between solely aragonitic and solely high-Mg calcitic stylasterids. ] in the CF would in turn translate to low CF Ω Aragonite (average 1.5 excluding S. ibericus; 4 corals have Ω Aragonite < 1). This contrasts with similar modelling of δ 11 B and B/Ca data in scleractinian corals which suggest CF Ω Aragonite is typically more than 5 times that of ambient seawater 38 . Although our estimates for stylasterid CF Ω Aragonite represent minimum values (i.e. no transport of Ca 2+ to the CF), it remains hard to explain how stylasterids commence calcification at all at such low saturation states. Like stylasterid corals, the δ 11 B of many foraminifera record the δ 11 B of borate and thus external seawater pH 35,39 , however microsensor studies show the pH of foraminiferal CF is actually slightly elevated (+ 0.5 pH units) 40 . We therefore explore alternative mechanisms that may facilitate mineralisation at low Ω and/or decouple the boron skeletal chemistry www.nature.com/scientificreports/ from calcification processes including: (i) bicarbonate active transport 41 , (ii) high boric acid to seawater flushing rates 25 , and (iii) organic molecule templating 42 . Despite cold-water scleractinian corals strongly upregulating pH, unusually low δ 11 B values (8.5‰) have been observed in their centres of calcification (or early mineralisation zones), particularly in Lophelia pertusa 41,43,44 . This disconnect between the internal pH and skeletal δ 11 B has been explained by bicarbonate (HCO 3 − ) active transport to the site of calcification 41 . The use of HCO 3 − as a source of DIC rather than CO 2 (from respiration and diffusion) promotes mineralisation as it releases fewer protons during conversion to CO 3 2− . The bicarbonate active transport model suggests that seawater borate is co-transported to the site of calcification, resulting in low skeletal δ 11 B values closer to seawater borate 41 . While this mechanism could explain low δ 11 B values in stylasterid corals, studies of the scleractinian coral Lophelia pertusa suggest that these centres of calcification are also associated with low B/Ca compared to the bulk coral (− 40%) 41,44 . This contrasts with the extremely high B/Ca values we observe in our aragonitic stylasterid corals compared to Scleractinia, suggesting an alternative mechanism is driving their boron skeletal chemistry.
Another approach to reconciling discrepancies between pH derived from microsensors and geochemistry comes from coral modelling work by Gagnon et al. 25 . In this model, internal scleractinian pH is held at a high, but constant, value of 8.9 (resulting in their characteristic high ∆δ 11 B) and low skeletal δ 11 B values are instead explained by higher rates of boric acid diffusion (D B ) relative to seawater exchange in the CF (kz; where k is the rate of seawater exchange and z is the volume to surface area ratio of the CF); where D B /kz scales linearly with seawater dissolved inorganic carbon ([DIC]). High D B /kz may therefore explain the low δ 11 B values in stylasterid corals, with low seawater flushing rates allowing them to occupy hostile growth environments (e.g. Ω Aragonite as low as 0.5 (depths > 500 m) in the high latitude North Pacific 45 ). To explore this hypothesis further, we use U/Ca and B/Ca ratios, with their sensitivity to the carbonate chemistry of seawater to test the potential role of D B /kz in stylasterids.
A positive correlation between carbonate δ 11 B and B/Ca is readily explained because at high pH more of the boron is in the borate ionic form and readily incorporated into the carbonate lattice 46 . This phenomenon has been observed in scleractinian corals 47 and crustose coralline algae 18,21 (Fig. 4). However, we find the opposite-a negative correlation between δ 11 B and B/Ca in sample replicates of stylasterid corals and particularly high B/Ca ratios in aragonitic specimens which cannot be explained by this effect. Although boron is preferentially incorporated into an aragonite lattice over calcite 48  The pH and/or [CO 3 2− ] dependency of uranyl ion complexation in seawater gives a strong theoretical basis for the sensitivity of coral U/Ca to the carbonate system 50,51 . The master variable however is not clear and many potential drivers of coral U/Ca have been suggested including: inverse relationships with (i) calcifying fluid [CO 3 2− ] 50,52,53 , (ii) pH 53,54 , and (iii) [DIC] 55 , as well as (iv) positive correlation with the rate of seawater replenishment of the coral calcifying space (kz) 55 . Evidence for the pH effect on U/Ca comes from positive covariance between δ 18 O and U/Ca 53 (Fig. 5A) and negative covariance between δ 11 B and U/Ca 47 (Fig. 5B) measured in replicate samples of scleractinian corals that strongly upregulate pH at the site of calcification. Thus while covariation of δ 18 O and δ 13 C ratios suggests perhaps some modification of internal pH 28 , the absence of correlation between δ 18 O and U/Ca (either between stylasterid coral specimens or between replicates) suggests that any pH upregulation in stylasterids is small enough not to impact U/Ca (Fig. 5A). Stylasterid coral results further contrast with scleractinian corals by exhibiting a positive correlation between δ 11 B and U/Ca between sample replicates (Fig. 5B). This is a counterintuitive result in the context of the pH and [CO 3 2− ] effects on coral U/Ca ratios (e.g. 53 ), however if D B /kz were the main driver of stylasterid coral U/Ca (e.g. 55 ) then the positive covariance between δ 11 B and U/Ca can be reconciled (Fig. 5B) along with low U/Ca values that negatively correlate with B/ Ca (Fig. 5C). Thus, high D B /kz can explain both the boron and uranium skeletal chemistry of stylasterids, with any small degree of biologically induced pH increase at the site of calcification (required to facilitate boric acid diffusion) potentially masked by low seawater flushing rates.
The mechanisms discussed so far have largely focussed on physiochemical processes inside a coral where mineralisation (ion-by-ion attachment) is dictated by Ω of the CF and where the elemental skeletal chemistry is driven by growth rate effects. Recent studies however highlight the important role that coral organic molecules may play in biocalcification (e.g. review by Drake et al. 42 ). Acid-rich proteins in corals can spontaneously catalyse the formation of amorphous CaCO 3 56 . Under this biologically mediated framework, the calcifying space requiring manipulation is just nanometers in size, comprising vesicles that both bind Ca 2+ and transport the amorphous CaCO 3 to the site of deposition 42 . This mechanism yields elemental skeletal chemistry which is more dependent on coral physiology 42 . The contention that stylasterid mineralogy (aragonite or high-Mg calcite) is more dictated by genetics rather than environmental parameters (e.g. seawater Ω Aragonite ) 29 does speak for a strong biological control on calcification. Thus, while aragonite may be vulnerable to dissolution 7 , it is potentially the more thermodynamically favourable mineral to form when mediated by acid-rich proteins in corals from modern seawater 56 . A reliance on organic matrix templates for mineralisation may therefore explain the paradoxically low skeletal δ 11 B values in stylasterids given their depth habitat. The presence of seawater at the site of calcification would also result in low skeletal δ 11 B values directly linked to ambient seawater pH. A strong biological control on calcification would therefore not preclude the use of stylasterid δ 11 B for use as a tracer for past seawater pH (e.g. Eq. (1); Supplementary information).
The energetic burden of calcifying via biologically-controlled organic molecule templating is considered low, such that it may confer resilience to organisms against future changes in ocean pH 56  www.nature.com/scientificreports/ the low pH and undersaturated habitats in which stylasterid corals appear to thrive are on seamounts 45 . These submarine features, particularly those at high-latitude, are often areas of localised current variability (suitable for larval dispersion), high primary production, and high organic flux to the seafloor (food supply), resulting in biodiversity hot-spots 57 . A ready source of metabolic energy is important in many marine calcifiers (e.g. 14 ), therefore it is possible that stylasterid corals compensate for low saturation conditions with additional metabolic energy sourced from an abundant food supply in these otherwise seemingly inhospitable environments. Some resilience to low pH waters may also come from their relatively thick layer of organic tissue that has been shown to shield the skeletons of other corals from undersaturated seawater 58 .

Conclusions
Stylasterid corals are of great ecological importance, occupying niches that other corals do not, thus providing vital habitat for other benthic organisms 26,59 . Understanding and mitigating the impacts of climate change on these corals is therefore key to their conservation. The low skeletal δ 11 B values that we record in all stylasterid corals, regardless of their mineralogy, closely resemble seawater δ 11 B borate suggesting that any biological upregulation of pH at the site of calcification in stylasterids is small. This style of biocalcification contrasts greatly with other aragonitic deep-water biocalcifiers (e.g. Scleractinia) and may be a result of an entirely different evolutionary history. For instance, many benthic marine fauna are thought to have originated in shallow waters and evolved to fill deep-water niches, whereas stylasterids are thought to have undergone an opposing, offshore to onshore, habitat expansion 60 . We argue that the low δ 11 B and generally high B/Ca and low U/Ca values we record in aragonitic stylasterids are best explained by (i) lower seawater exchange rates and/or higher rates of boric acid diffusion into the CF compared to scleractinian corals or (ii) a reliance on organic matrix templating for calcification. Without a strong internal pH upregulating mechanism, physiochemical models of biocalcification would predict that stylasterids precipitating aragonite will be more vulnerable to future declines in seawater pH than those that have adopted the more dissolution-resistant high-Mg calcitic mineral form. However, if their calcification is strongly biologically mediated, this strategy may prove advantageous over other taxa which rely on ion pumping to modify their calcifying fluid chemistry (e.g. scleractinian corals). Determining the energetic burden of their calcification strategy will therefore be key to understanding the resilience of stylasterids to future change.  28,29,61 . The Li/Mg and Sr/Ca ratios of these same specimens were previously measured by Stewart et al. 61 . Because E. laterorifa Mg/ Ca values are less than 140 µmol/mol, the high-Mg calcitic corals in this study are considered theoretically more resistant to dissolution than pure aragonitic counterparts based solely on their mineralogy 7 .

Methods
Matching hydrographic data. To characterise ambient seawater conditions at each site we use the same paired hydrographic data to that of Stewart et al. 61 . Alkalinity and [DIC] measurements were used in conjunction with temperature, salinity, and nutrient data to calculate full carbonate system parameters at each location including pH (total scale); Seacarb package in R 62 ). This calculation used carbonate dissociation constants from Lueker et al. 63 and the boron to salinity ratios of Lee et al. 64 . Where possible seawater hydrographic data were taken from co-located remotely operated vehicle or CTD (conductivity, temperature, and depth) profiles taken during the respective research cruise (10 samples from cruises DY081, JC094, NBP1103, and NBP0805). Where paired shipboard data were unavailable results from nearby bottle data in the GLODAP v2 database were used 65 . Hydrographic stations were typically within ~ 150 km of coral sampling sites and 30 m depth of the samples. The maximum offset between corals and water data was at the Galapagos site (530 km distance and 89 m water depth) owing to the poor regional sampling coverage. To assess uncertainty on pH estimates caused by both true in-situ variability and distance between coral and hydrographic stations, a further two nearby bottle seawater measurements were selected from the combined GLODAP v2 and research cruise databases (Supplementary Information). For most sites this variability was within ± 0.04 pH units (2σ) with a maximum variation of ± 0.09 pH units. At sites where pH was more variable the most proximal bottle data is still preferred over averaging all three pH estimates. increase as a function of seawater pH 46,66,67 . The incorporation of this charged ion into marine calcium carbonates as they precipitate therefore forms the premise of the boron isotope pH-proxy 66 . Here we calculate δ 11 B borate of ambient seawater experienced by each coral using the seawater pH estimates above and the simplified equation (Eq. 4) rearranged from Zeebe and Wolf-Gladrow 67 : (4) δ 11 B borate = δ 11 B sw + (δ 11 B sw − 1000(α B − 1))10 pK * B −pH  68 , αB (1.027) is the fractionation factor between boric acid and the borate ion 69 , and pK B * is the dissociation constant of the two boron species calculated using site specific temperature, salinity, and pressure estimates (8.83 at 3 °C, 35 psu, and 700 m respectively, typical of sites in this study). Factoring for variation in temperature and salinity on pK B * , estimates of seawater pH uncertainty outlined above correspond to uncertainties on δ 11 B borate that are typically better than ± 0.15‰ (2 SD; maximum ± 0.57‰). Coral sample preparation. Coral samples for this study were air dried following collection. The majority of organic matter was removed through both physical scraping and treatment in dilute NaClO for 12 h. Cross sectional discs (~ 2 mm thickness) were cut using a rotary cutting tool from the central trunk (or widest branch) of stylasterid samples. Microstructures within deep-sea corals are known to exhibit contrasting boron concentration 47 and isotopic composition 43 . Similarly, the δ 18 O composition of growing tips of stylasterid branches has been shown to be further from seawater equilibrium than bulk samples 28 , hence apical tips were avoided and large samples were cut (~ 50 mg; i.e. × 10 mass required for analysis) to diminish the influence of a single microstructural component. To confirm this, corals were also sampled and analysed in duplicate to characterise variability between solid pieces cut from the same coral.

Estimation of internal Ω
Coral fragments were finely crushed using a pestle and mortar before 5 to 10 mg of the homogenous powder was taken. Residual organic matter was removed using warm 1% H 2 O 2 (80 °C; buffered in NH 4 OH) and a weak acid leach (0.0005 M HNO 3 ) before powders were dissolved in distilled 0.5 M HNO 3 .
Analytical techniques. All analyses were performed at the University of Bristol. An aliquot of the dissolved sample was analysed by ICP-MS using well-characterised, matrix-matched, synthetic standard solutions to give B/Ca and U/Ca ratios. Samples and standards were introduced in 0.5 M HNO 3 and a 0.5 M HNO 3 and 0.3 M HF acid wash solution was utilised between samples/standards to aid B wash out 31 . Repeat analysis of NIST RM 8301 (Coral) yielded mean B/Ca and U/Ca ratios of 527 ± 2% and 822 ± 1% µmol/mol (RSD; n = 35) which are within analytical uncertainty of the interlaboratory consensus value for this reference material 70 .
An aliquot of the dissolved sample containing ~ 25 ng B was separated from the carbonate matrix using 20 μl micro-columns containing Amberlite IRA 743 boron-specific anionic exchange resin 71 . The δ 11 B of purified boron samples were measured by Multi-Collector ICP-MS against NIST SRM 951 35,71 . In this case, all samples, blanks, and standard solutions were introduced to the instrument in a 0.5 M HNO 3 and 0.3 M HF acid matrix again to ensure optimal B wash out 31 . Full procedural uncertainty was assessed using repeat measurement of NIST RM 8301 (Coral) yielding an average δ 11 B value of 24.27 ± 0.16‰ (n = 25; 2 s) which again was within uncertainty of interlaboratory consensus values 70 . Total procedural blanks (n = 6) were small (less than 39 ng of boron) and were thus < 0.2% of the boron loaded onto columns for samples.